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Abstract:

The present invention provides methods and compositions for identifying
subjects at risk of developing a complication of pregnancy, such as
preeclampsia or preterm labor. The compositions are microRNAs and
associated nucleic acids.

Claims:

1.-20. (canceled)

21. A method for determining whether a pregnant female subject is at risk
of developing preeclampsia, said method comprising: obtaining a
biological test sample from said female subject and a control biological
sample from a pregnant woman of same gestational age with no clinical or
laboratory evidence of hypertensive disorder of pregnancy; extracting RNA
from the test biological sample and the control biological sample;
comparing the expression profile of a nucleic acid sequence selected from
the group consisting of SEQ ID NOS: 5-16, a fragment thereof and a
sequence having at least about 80% identical thereto in the biological
sample and the control biological sample; determining whether said
pregnant female is at risk of developing preeclampsia based on increased
expression of said nucleic acid sequence in the test biological sample
relative to the control biological sample; and determining a treatment
strategy for said pregnant female based on the increased expression of
said nucleic acid in the test biological sample relative to the control
biological sample.

22. The method of claim 21, wherein said biological sample is selected
from the group consisting of bodily fluid and a tissue sample.

31. A method for detecting or monitoring preterm labor in a pregnant
female subject, said method comprising comparing the expression profile
of a nucleic acid sequence selected from the group consisting of SEQ ID
NOS: 18-25, a fragment thereof and sequence having at least about 80%
identity thereto in a biological sample from the subject to a
predetermined standard expression profile, wherein a significant
difference in the expression profile of said nucleic acid sequence in the
sample as compared to a predetermined expression profile standard
indicates that the subject is in preterm labor.

32. The method of claim 31, wherein the predetermined standard expression
profile corresponds to the expression profile of said nucleic acid
sequence in a pregnant female subject who is not at risk of preterm
labor.

33. A kit for determining if a female subject is at risk of developing
preeclampsia, said kit comprising a probe comprising a nucleic acid
sequence that is complementary to a sequence selected from the group
consisting of SEQ ID NOS: 5-17, a fragment thereof and a sequence having
at least about 80% identity thereto.

34. A kit for determining if a female subject is at risk of having a
preterm labor, said kit comprising a probe comprising a nucleic acid
sequence that is complementary to a sequence selected from the group
consisting of SEQ ID NOS: 18-25,o fragment thereof and a sequence having
at least about 80% identity thereto.

35. The kit of claim 33, wherein the kit further comprises forward and
reverse primers.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C.
§119(e) to U.S. Provisional Application No. 61/023,859, filed Jan.
27, 2008; U.S. and U.S. Provisional Application No. 61/040,671, filed
Mar. 30, 2008 which are herein incorporated by reference in their
entirety.

FIELD OF THE INVENTION

[0002] The invention relates to methods and compositions for identifying
subjects at risk of developing a complication of pregnancy, such as
preeclampsia or preterm labor. The compositions are microRNAs and
associated nucleic acids.

BACKGROUND OF THE INVENTION

[0003] Circulating nucleic acids (CNAs) in body fluids offer unique
opportunities for early diagnosis of clinical conditions. Specific
clinical biomarkers have the potential to revolutionize diagnosis and
treatment of various medical conditions, such as abnormal pregnancies.
The challenge of diverse biomedical research fields has been to identify
biomarkers in body fluids, such as serum. In recent years it has become
clear that both cell-free DNA and mRNA are present in serum, as well as
in other body fluids, and represent potential biomarkers. However,
monitoring the typically small amounts of these CNAs in body fluids
requires sensitive detection methods, which are not currently clinically
applicable.

[0004] microRNAs (miRNAs, miRs) have emerged as an important novel class
of regulatory RNA, which has profound impact on a wide array of
biological processes. These small (typically 17-24 nucleotides long)
non-coding RNA molecules can modulate protein expression patterns by
promoting RNA degradation, inhibiting mRNA translation, and also
affecting gene transcription. miRs play pivotal roles in diverse
processes such as development and differentiation, control of cell
proliferation, stress response and metabolism. There are currently about
850 known human miRs.

[0005] Preeclampsia, complicating 3-5% of pregnancies, is associated with
substantial risks for both the mother and the fetus. Although many
theories exist for the etiology and pathogenesis of preeclampsia, its
direct etiology remains unidentified. There has been little progress in
the treatment of this disorder; the cure remains delivery of the fetus
and removal of the placenta.

[0006] Effective management strategies for identifying and treating
preterm labor are required to prevent preterm birth. Early births
resulting from preterm labor result in a heavy burden of infant mortality
and morbidity. Preterm birth is a factor in three-quarters of neonatal
mortality and one-half of long-term neurologic impairment in children.

[0007] Early detection and management of preterm labor helps to prevent
preterm birth and its potential neonatal sequelae, which include
respiratory distress syndrome, sepsis, intraventricular hemorrhage,
necrotizing enterocolitis, patent ductus arteriosus, and
hyperbilirubinemia; however, widespread treatment of women with signs and
symptoms of preterm labor has not significantly reduced the prevalence of
preterm birth, underscoring the need to improve current methods for
detecting preterm labor.

[0008] There is an unmet need for a reliable method for identifying
subjects at risk of developing a complication of pregnancy, such as
preeclampsia or preterm labor.

SUMMARY OF THE INVENTION

[0009] The present invention demonstrates for the first time that
circulating microRNAs are novel serum markers with high stability and
signature robustness.

[0010] The invention provides a method of determining a physiological
condition in a subject, said method comprising detecting the level of a
microRNA in a serum sample obtained from the subject, wherein a level of
the microRNA different from a control is indicative of said physiological
condition in said subject. According to some embodiments the
physiological condition is a pregnancy-associated disorder. According to
other embodiments the pregnancy-associated disorder is preeclampsia or
preterm labor. According to some embodiments the detection of the
microRNA level is determined by real-time PCR.

[0011] The invention further provides specific nucleic acid sequences that
may be used for the identification and diagnosis of a complication of
pregnancy, such as preeclampsia or preterm labor. According to some
embodiments said nucleic acid sequences are selected from the group
consisting of SEQ ID NOS: 1-110, a fragment thereof and a sequence having
at least about 80% identity thereto.

[0012] The invention further provides a method for determining or aiding
in the determination that a female subject is at risk of developing
preeclampsia, comprising comparing the expression profile of a nucleic
acid sequence selected from the group consisting of SEQ ID NOS: 5-17, a
fragment thereof and a sequence having at least about 80% identity
thereto in a biological sample from the subject to be assessed for risk
of developing preeclampsia to a predetermined standard expression
profile, wherein a significant difference in expression profile of said
nucleic acid sequence in the sample as compared to the predetermined
standard expression profile indicates that the subject is at risk of
developing preeclampsia.

[0013] According to some embodiments, the predetermined standard
expression profile corresponds to the expression profile of said nucleic
acid sequence in a pregnant female subject who is not at risk of
developing preeclampsia.

[0014] According to other embodiments, said biological sample is selected
from the group consisting of bodily fluid and a tissue sample. According
to some embodiments, said tissue is a fresh, frozen, fixed, wax-embedded
or formalin fixed paraffin-embedded (FFPE) tissue.

[0015] According to one embodiment, the tissue sample is placenta sample
or uterine myometrium sample. According to some embodiments, said bodily
fluid sample is serum sample.

[0016] According to some embodiments, the method comprising determining
the expression profile of at least two nucleic acid sequences. According
to some embodiments the method further comprising combining one or more
expression ratios. According to some embodiments, the expression levels
are determined by a method selected from the group consisting of nucleic
acid hybridization and nucleic acid amplification. According to some
embodiments, the nucleic acid hybridization is performed using a
solid-phase nucleic acid biochip array. According to certain embodiments,
the nucleic acid hybridization is performed using in situ hybridization.
According to other embodiments, the nucleic acid amplification method is
real-time PCR. According to one embodiment, said real-time PCR is
quantitative real-time PCR (qRT-PCR).

[0017] The invention further provides a method for detecting or monitoring
a preterm labor in a female subject, comprising comparing the expression
profile of a nucleic acid sequence selected from the group consisting of
SEQ ID NOS: 18-25, a fragment thereof and a sequence having at least
about 80% identity thereto in a biological sample from the subject to a
predetermined standard expression profile, wherein a significant
difference in the expression profile of said nucleic acid sequence in the
sample as compared to the predetermined standard expression profile
indicates that the subject is in preterm labor.

[0018] According to some embodiments, the predetermined standard
expression profile corresponds to the expression profile of said nucleic
acid sequence in a pregnant female subject who is not at risk of preterm
labor.

[0019] The invention further provides a kit for determining if a subject
female is at risk of developing preeclampsia, said kit comprises a probe
comprising a nucleic acid sequence that is complementary to a sequence
selected from the group consisting of SEQ ID NOS: 5-17, a fragment
thereof and a sequence having at least about 80% identical thereto.

[0020] The invention further provides a kit for determining if a female
subject is at risk of having a preterm labor, said kit comprising a probe
comprising a nucleic acid sequence that is complementary to a sequence
selected from the group consisting of SEQ ID NOS: 18-25, a fragment
thereof and a sequence having at least about 80% identity thereto.

[0021] According to some embodiments, the kit further comprises forward
and reverse primers.

[0022] These and other embodiments of the present invention will become
apparent in conjunction with the figures, description and claims that
follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows differential expression of four microRNAs in the sera
of pregnant vs. non-pregnant women. Expression level is specified as
50-CT, where CT is the cycle threshold of the PCR reaction.
Results were normalized by subtracting the global microRNA expression in
the sample (average CT of the 6 microRNAs chosen for normalization)
from the CT level of each microRNA.

[0026] C)
"Pregnancy classification" according to the levels of three microRNAs in
the sera of pregnant vs. non-pregnant women. Discrimination of pregnant
women from non-pregnant women based on microRNA expression levels in
their sera. Circles represent non-pregnant women, triangles represent
pregnant women and dots represent 3rd trimester. The location of
each symbol in the plot represents the collective expression of all three
microRNAs in a given serum. The y axis indicates the expression level of
mir 527, and the x axis indicates the average expression level of
miR-520d-5p and miR-526a.

[0037] The invention is based on the discovery that specific nucleic acid
sequences (SEQ ID NOS: 1-110) may be used for the identification and
diagnosis of a complication of pregnancy, such as preeclampsia or preterm
labor. The present invention demonstrates for the first time that serum
levels of particular microRNAs may serve as diagnostic biomarkers for
diverse physiological and pathological conditions. Moreover, the present
invention demonstrates the ease and reliability of determining body fluid
microRNA profiles and thus, paves the way for their wide application,
both in the research laboratory and in the clinic. The methods of the
present invention have high sensitivity and specificity.

DEFINITIONS

[0038] Before the present compositions and methods are disclosed and
described, it is to be understood that the terminology used herein is for
the purpose of describing particular embodiments only and is not intended
to be limiting. It must be noted that, as used in the specification and
the appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.

[0039] For the recitation of numeric ranges herein, each intervening
number there between with the same degree of precision is explicitly
contemplated. For example, for the range of 6-9, the numbers 7 and 8 are
contemplated in addition to 6 and 9, and for the range 6.0-7.0, the
number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are
explicitly contemplated.

[0040] About

[0041] As used herein, the term "about" refers to +/-10%.

[0042] Antisense

[0043] The term "antisense," as used herein, refers to nucleotide
sequences which are complementary to a specific DNA or RNA sequence. The
term "antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules may be
produced by any method, including synthesis by ligating the gene(s) of
interest in a reverse orientation to a viral promoter which permits the
synthesis of a complementary strand. Once introduced into a cell, this
transcribed strand combines with natural sequences produced by the cell
to form duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant phenotypes may be
generated.

[0044] Attached

[0045] "Attached" or "immobilized" as used herein to refer to a probe and
a solid support may mean that the binding between the probe and the solid
support is sufficient to be stable under conditions of binding, washing,
analysis, and removal. The binding may be covalent or non-covalent.
Covalent bonds may be formed directly between the probe and the solid
support or may be formed by a cross linker or by inclusion of a specific
reactive group on either the solid support or the probe or both
molecules. Non-covalent binding may be one or more of electrostatic,
hydrophilic, and hydrophobic interactions. Included in non-covalent
binding is the covalent attachment of a molecule, such as streptavidin,
to the support and the non-covalent binding of a biotinylated probe to
the streptavidin. Immobilization may also involve a combination of
covalent and non-covalent interactions.

[0046] Biological Sample

[0047] "Biological sample" as used herein may mean a sample of biological
tissue or fluid that comprises nucleic acids. Such samples include, but
are not limited to, tissue or fluid isolated from animals. Biological
samples may also include sections of tissues such as biopsy and autopsy
samples, FFPE samples, frozen sections taken for histologic purposes,
blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin.
Biological samples also include explants and primary and/or transformed
cell cultures derived from animal or patient tissues. Biological samples
may also be blood, a blood fraction, urine, effusions, ascitic fluid,
saliva, cerebrospinal fluid, cervical secretions, vaginal secretions,
endometrial secretions, gastrointestinal secretions, bronchial
secretions, sputum, cell line, tissue sample, or secretions from the
breast. A biological sample may be provided by removing a sample of cells
from an animal, but can also be accomplished by using previously isolated
cells (e.g., isolated by another person, at another time, and/or for
another purpose), or by performing the methods described herein in vivo.
Archival tissues, such as those having treatment or outcome history, may
also be used.

[0048] Complement

[0049] "Complement" or "complementary" as used herein refer to a nucleic
acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base
pairing between nucleotides or nucleotide analogs of nucleic acid
molecules. A full complement or fully complementary may mean 100%
complementary base pairing between nucleotides or nucleotide analogs of
nucleic acid molecules.

[0050] Ct

[0051] Ct signals represent the first cycle of PCR where amplification
crosses a threshold (cycle threshold) of fluorescence. Accordingly, low
values of Ct represent high abundance or expression levels of the
microRNA. In some embodiments the PCR Ct signal is normalized such that
the normalized Ct remains inversed from the expression level. In other
embodiments the PCR Ct signal may be normalized and then inverted such
that low normalized-inverted Ct represents low abundance or expression
levels of the microRNA.

[0052] Detection

[0053] "Detection" may mean detecting the presence of a component in a
sample. Detection may also mean detecting the absence of a component.
Detection may also mean measuring the level of a component, either
quantitatively or qualitatively.

[0054] Differential Expression

[0055] "Differential expression" may mean qualitative or quantitative
differences in the temporal and/or cellular gene expression patterns
within and among cells and tissue. Thus, a differentially expressed gene
may qualitatively have its expression altered, including an activation or
inactivation, in, e.g., normal versus disease tissue. Genes may be turned
on or turned off in a particular state, relative to another state thus
permitting comparison of two or more states. A qualitatively regulated
gene may exhibit an expression pattern within a state or cell type which
may be detectable by standard techniques. Some genes may be expressed in
one state or cell type, but not in both. Alternatively, the difference in
expression may be quantitative, e.g., in that expression is modulated,
either up-regulated, resulting in an increased amount of transcript, or
down-regulated, resulting in a decreased amount of transcript. The degree
to which expression differs need only be large enough to quantify via
standard characterization techniques such as expression arrays,
quantitative reverse transcriptase PCR, northern analysis, real-time PCR,
and RNase protection.

[0056] Ectopic Pregnancy

[0057] An "ectopic pregnancy" refers to an abnormal pregnancy in which a
fertilized egg has implanted outside the uterus. Although in most cases
of ectopic pregnancy the egg settles in the fallopian tubes, this term
also encompasses abnormal pregnancies where the fertilized egg is
implanted in a woman's ovary, abdomen, or cervix.

[0058] Expression Profile

[0059] The term "expression profile" is used broadly to include a genomic
expression profile, e.g., an expression profile of microRNAs. Profiles
may be generated by any convenient means for determining a level of a
nucleic acid sequence e.g. quantitative hybridization of microRNA,
labeled microRNA, amplified microRNA, cRNA, etc., quantitative PCR, ELISA
for quantitation, and the like, and allow the analysis of differential
gene expression between two samples. A subject or patient sample, e.g.,
cells or a collection thereof, e.g., tissues, is assayed. Samples are
collected by any convenient method, as known in the art. Nucleic acid
sequences of interest are nucleic acid sequences that are found to be
predictive, including the nucleic acid sequences provided above, where
the expression profile may include expression data for 2, 5, 10, 20, 25,
50, 100 or more of, including all of the listed nucleic acid sequences.
According to some embodiments, the term "expression profile" means
measuring the abundance of the nucleic acid sequences in the measured
samples.

[0060] Expression Ratio

[0061] "Expression ratio" as used herein refers to relative expression
levels of two or more nucleic acids as determined by detecting the
relative expression levels of the corresponding nucleic acids in a
biological sample.

[0062] Fragment

[0063] "Fragment" is used herein to indicate a non-full length part of a
nucleic acid. Thus, a fragment is itself also a nucleic acid.

[0064] Gene

[0065] "Gene" used herein may be a natural (e.g., genomic) or synthetic
gene comprising transcriptional and/or translational regulatory sequences
and/or a coding region and/or non-translated sequences (e.g., introns,
5'- and 3'-untranslated sequences). The coding region of a gene may be a
nucleotide sequence coding for an amino acid sequence or a functional
RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA. A
gene may also be an mRNA or cDNA corresponding to the coding regions
(e.g., exons and miRNA) optionally comprising 5'- or 3'-untranslated
sequences linked thereto. A gene may also be an amplified nucleic acid
molecule produced in vitro comprising all or a part of the coding region
and/or 5'- or 3'-untranslated sequences linked thereto.

[0066] Groove Binder/Minor Groove Binder (MGB)

[0067] "Groove binder" and/or "minor groove binder" may be used
interchangeably and refer to small molecules that fit into the minor
groove of double-stranded DNA, typically in a sequence-specific manner.
Minor groove binders may be long, flat molecules that can adopt a
crescent-like shape and thus, fit snugly into the minor groove of a
double helix, often displacing water. Minor groove binding molecules may
typically comprise several aromatic rings connected by bonds with
torsional freedom such as furan, benzene, or pyrrole rings. Minor groove
binders may be antibiotics such as netropsin, distamycin, berenil,
pentamidine and other aromatic diamidines, Hoechst 33258, SN 6999,
aureolic anti-tumor drugs such as chromomycin and mithramycin, CC-1065,
dihydrocyclopyrroloindole tripeptide (DPI3),
1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3), and
related compounds and analogues, including those described in Nucleic
Acids in Chemistry and Biology, 2d ed., Blackburn and Gait, eds., Oxford
University Press, 1996, and PCT Published Application No. WO 03/078450,
the contents of which are incorporated herein by reference. A minor
groove binder may be a component of a primer, a probe, a hybridization
tag complement, or combinations thereof. Minor groove binders may
increase the of the primer or a probe to which they are attached,
allowing such primers or probes to effectively hybridize at higher
temperatures.

[0068] Identity

[0069] "Identical" or "identity" as used herein in the context of two or
more nucleic acids or polypeptide sequences may mean that the sequences
have a specified percentage of residues that are the same over a
specified region. The percentage may be calculated by optimally aligning
the two sequences, comparing the two sequences over the specified region,
determining the number of positions at which the identical residue occurs
in both sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
specified region, and multiplying the result by 100 to yield the
percentage of sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered ends
and the specified region of comparison includes only a single sequence,
the residues of single sequence are included in the denominator but not
the numerator of the calculation. When comparing DNA and RNA, thymine (T)
and uracil (U) may be considered equivalent. Identity may be performed
manually or by using a computer sequence algorithm such as BLAST or BLAST
2.0.

[0070] Label

[0071] "Label" as used herein may mean a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, chemical, or
other physical means. For example, useful labels include 32P,
fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly
used in an ELISA), biotin, digoxigenin, or haptens and other entities
which can be made detectable. A label may be incorporated into nucleic
acids and proteins at any position.

[0072] Nucleic Acid

[0073] "Nucleic acid" or "oligonucleotide" or "polynucleotide" used herein
may mean at least two nucleotides covalently linked together. The
depiction of a single strand also defines the sequence of the
complementary strand. Thus, a nucleic acid also encompasses the
complementary strand of a depicted single strand. Many variants of a
nucleic acid may be used for the same purpose as a given nucleic acid.
Thus, a nucleic acid also encompasses substantially identical nucleic
acids and complements thereof. A single strand provides a probe that may
hybridize to a target sequence under stringent hybridization conditions.
Thus, a nucleic acid also encompasses a probe that hybridizes under
stringent hybridization conditions.

[0074] Nucleic acids may be single stranded or double stranded, or may
contain portions of both double stranded and single stranded sequence.
The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid,
where the nucleic acid may contain combinations of deoxyribo- and
ribo-nucleotides, and combinations of bases including uracil, adenine,
thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine
and isoguanine. Nucleic acids may be obtained by chemical synthesis
methods or by recombinant methods.

[0075] A nucleic acid will generally contain phosphodiester bonds,
although nucleic acid analogs may be included that may have at least one
different linkage, e.g., phosphoramidate, phosphorothioate,
phosphorodithioate, or O-methylphosphoroarnidite linkages and peptide
nucleic acid backbones and linkages. Other analog nucleic acids include
those with positive backbones; non-ionic backbones, and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033 and
5,034,506, which are incorporated by reference. Nucleic acids containing
one or more non-naturally occurring or modified nucleotides are also
included within one definition of nucleic acids. The modified nucleotide
analog may be located for example at the 5'-end and/or the 3'-end of the
nucleic acid molecule. Representative examples of nucleotide analogs may
be selected from sugar- or backbone-modified ribonucleotides. It should
be noted, however, that also nucleobase-modified ribonucleotides, i.e.
ribonucleotides, containing a non-naturally occurring nucleobase instead
of a naturally occurring nucleobase such as uridines or cytidines
modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo
uridine; adenosines and guanosines modified at the 8-position, e.g.
8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and
N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The
2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH,
SR, NH2, NHR, NR2 or CN, wherein R is C1-C6 alkyl,
alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also
include nucleotides conjugated with cholesterol through, e.g., a
hydroxyprolinol linkage as described in Krutzfeldt et al., Nature
438:685-689 (2005), Soutschek et al., Nature 432:173-178 (2004), and U.S.
Patent Publication No. 20050107325, which are incorporated herein by
reference. Additional modified nucleotides and nucleic acids are
described in U.S. Patent Publication No. 20050182005, which is
incorporated herein by reference. Modifications of the ribose-phosphate
backbone may be done for a variety of reasons, e.g., to increase the
stability and half-life of such molecules in physiological environments,
to enhance diffusion across cell membranes, or as probes on a biochip.
The backbone modification may also enhance resistance to degradation,
such as in the harsh endocytic environment of cells. The backbone
modification may also reduce nucleic acid clearance by hepatocytes, such
as in the liver and kidney. Mixtures of naturally occurring nucleic acids
and analogs may be made; alternatively, mixtures of different nucleic
acid analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made.

[0076] Pregnancy-Associated Disorder

[0077] The term "pregnancy-associated disorder" as used in this
application, refers to any condition or disease that may affect a
pregnant woman, the fetus the woman is carrying, or both the woman and
the fetus. Such a condition or disease may manifest its symptoms during a
limited time period, e.g., during pregnancy or delivery, or may last the
entire life span of the fetus following its birth. Some examples of a
pregnancy-associated disorder include preeclampsia, preterm labor,
ectopic pregnancy, and fetal chromosomal abnormalities.

[0078] Preeclampsia

[0079] The term "preeclampsia" as used herein refers to a condition that
occurs during pregnancy, the main symptom of which is various forms of
high blood pressure often accompanied by the presence of proteins in the
urine and edema (swelling). Preeclampsia, sometimes called toxemia of
pregnancy, is related to a more serious disorder called "eclampsia,"
which is preeclampsia together with seizures. These conditions usually
develop during the second half of pregnancy (after 20 weeks), though they
may develop shortly after birth or before 20 weeks of pregnancy.

[0080] Preterm Labor

[0081] The term "preterm labor" or "premature labor" as used herein refers
to the condition where labor that begins more than three weeks before the
full gestation period of about 40 weeks, which often leads to premature
birth if not treated.

[0082] Probe

[0083] "Probe" as used herein may mean an oligonucleotide capable of
binding to a target nucleic acid of complementary sequence through one or
more types of chemical bonds, usually through complementary base pairing,
usually through hydrogen bond formation. Probes may bind target sequences
lacking complete complementarity with the probe sequence depending upon
the stringency of the hybridization conditions. There may be any number
of base pair mismatches which will interfere with hybridization between
the target sequence and the single stranded nucleic acids described
herein. However, if the number of mutations is so great that no
hybridization can occur under even the least stringent of hybridization
conditions, the sequence is not a complementary target sequence. A probe
may be single stranded or partially single and partially double stranded.
The strandedness of the probe is dictated by the structure, composition,
and properties of the target sequence. Probes may be directly labeled or
indirectly labeled such as with biotin to which a streptavidin complex
may later bind.

[0084] Reference Expression Profile

[0085] As used herein, the phrase "reference expression profile" or
"predetermined standard expression profile" refers to a criterion
expression value to which measured values are compared in order to
determine the detection of a subject at risk of developing a complication
of pregnancy. The reference expression profile may be based on the
abundance of the nucleic acids, or may be based on a combined metric
score thereof.

[0086] Stringent Hybridization Conditions

[0087] "Stringent hybridization conditions" used herein may mean
conditions under which a first nucleic acid sequence (e.g., probe) will
hybridize to a second nucleic acid sequence (e.g., target), such as in a
complex mixture of nucleic acids. Stringent conditions are
sequence-dependent and will be different in different circumstances.
Stringent conditions may be selected to be about 5-10° C. lower
than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength pH. The Tm may be the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50% of
the probes complementary to the target hybridize to the target sequence
at equilibrium (as the target sequences are present in excess, at
Tm, 50% of the probes are occupied at equilibrium). Stringent
conditions may be those in which the salt concentration is less than
about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration
(or other salts) at pH 7.0 to 8.3 and the temperature is at least about
30° C. for short probes (e.g., about 10-50 nucleotides) and at
least about 60° C. for long probes (e.g., greater than about 50
nucleotides). Stringent conditions may also be achieved with the addition
of destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal may be at least 2 to 10 times background
hybridization. Exemplary stringent hybridization conditions include the
following: 50% formamide, 5×SSC, and 1% SDS, incubating at
42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with
wash in 0.2×SSC, and 0.1% SDS at 65° C.

[0091] "Substantially identical" used herein may mean that a first and
second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with
respect to nucleic acids, if the first sequence is substantially
complementary to the complement of the second sequence.

[0092] Subject

[0093] As used herein, the term "subject" refers to a mammal, including
both human and other mammals. The methods of the present invention are
preferably applied to human subjects.

[0094] Target Nucleic Acid

[0095] "Target nucleic acid" as used herein may mean a nucleic acid or
variant thereof that may be bound by another nucleic acid. A target
nucleic acid may be a DNA sequence. The target nucleic acid may be an
RNA. The target nucleic acid may comprise a mRNA, tRNA, shRNA, siRNA or
Piwi-interacting RNA, or a pri-miRNA, pre-miRNA, miRNA, or anti-miRNA.
The target nucleic acid may comprise a target miRNA binding site or a
variant thereof. One or more probes may bind the target nucleic acid. The
target binding site may comprise 5-100 or 10-60 nucleotides. The target
binding site may comprise a total of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-40, 40-50,
50-60, 61, 62 or 63 nucleotides. The target site sequence may comprise at
least 5 nucleotides of the sequence of a target miRNA binding site
disclosed in U.S. patent application Ser. Nos. 11/384,049, 11/418,870 or
11/429,720, the contents of which are incorporated herein.

[0096] Tissue Sample

[0097] As used herein, a tissue sample is tissue obtained from a tissue
biopsy using methods well known to those of ordinary skill in the related
medical arts. Methods for obtaining the sample from the biopsy include
gross apportioning of a mass, microdissection, laser-based
microdissection, or other art-known cell-separation methods.

[0098] Variant

[0099] "Variant" used herein to refer to a nucleic acid may mean (i) a
portion of a referenced nucleotide sequence; (ii) the complement of a
referenced nucleotide sequence or portion thereof; (iii) a nucleic acid
that is substantially identical to a referenced nucleic acid or the
complement thereof; or (iv) a nucleic acid that hybridizes under
stringent conditions to the referenced nucleic acid, complement thereof,
or a sequence substantially identical thereto.

[0100] Wild Type

[0101] As used herein, the term "wild type" sequence refers to a coding,
non-coding or interface sequence is an allelic form of sequence that
performs the natural or normal function for that sequence. Wild type
sequences include multiple allelic forms of a cognate sequence, for
example, multiple alleles of a wild type sequence may encode silent or
conservative changes to the protein sequence that a coding sequence
encodes.

[0102] Considering the central role of microRNAs in development and
disease, the present invention highlights the medically relevant
potential of determining microRNA levels in serum and other body fluids.
Thus, microRNAs are a new class of CNAs that promise to serve as useful
clinical biomarker.

[0103] microRNA Processing

[0104] A gene coding for a miRNA may be transcribed leading to production
of an miRNA precursor known as the pri-miRNA. The pri-miRNA may be part
of a polycistronic RNA comprising multiple pri-miRNAs. The pri-miRNA may
form a hairpin with a stem and loop. The stem may comprise mismatched
bases.

[0105] The hairpin structure of the pri-miRNA may be recognized by Drosha,
which is an RNase III endonuclease. Drosha may recognize terminal loops
in the pri-miRNA and cleave approximately two helical turns into the stem
to produce a 60-70 nt precursor known as the pre-miRNA. Drosha may cleave
the pri-miRNA with a staggered cut typical of RNase III endonucleases
yielding a pre-miRNA stem loop with a 5' phosphate and ˜2
nucleotide 3' overhang. Approximately one helical turn of stem (˜10
nucleotides) extending beyond the Drosha cleavage site may be essential
for efficient processing. The pre-miRNA may then be actively transported
from the nucleus to the cytoplasm by Ran-GTP and the export receptor
Ex-portin-5.

[0106] The pre-miRNA may be recognized by Dicer, which is also an RNase
III endonuclease. Dicer may recognize the double-stranded stem of the
pre-miRNA. Dicer may also recognize the 5' phosphate and 3' overhang at
the base of the stem loop. Dicer may cleave off the terminal loop two
helical turns away from the base of the stem loop leaving an additional
5' phosphate and ˜2 nucleotide 3' overhang. The resulting
siRNA-like duplex, which may comprise mismatches, comprises the mature
miRNA and a similar-sized fragment known as the miRNA*. The miRNA and
miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA.
MiRNA* sequences may be found in libraries of cloned miRNAs but typically
at lower frequency than the miRNAs.

[0107] Although initially present as a double-stranded species with
miRNA*, the miRNA may eventually become incorporated as a single-stranded
RNA into a ribonucleoprotein complex known as the RNA-induced silencing
complex (RISC). Various proteins can form the RISC, which can lead to
variability in specificity for miRNA/miRNA* duplexes, binding site of the
target gene, activity of miRNA (repress or activate), and which strand of
the miRNA/miRNA* duplex is loaded in to the RISC.

[0108] When the miRNA strand of the miRNA:miRNA* duplex is loaded into the
RISC, the miRNA* may be removed and degraded. The strand of the
miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose
5' end is less tightly paired. In cases where both ends of the
miRNA:miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA*
may have gene silencing activity.

[0109] The RISC may identify target nucleic acids based on high levels of
complementarity between the miRNA and the mRNA, especially by nucleotides
2-7 of the miRNA. Only one case has been reported in animals where the
interaction between the miRNA and its target was along the entire length
of the miRNA. This was shown for mir-196 and Hox B8 and it was further
shown that mir-196 mediates the cleavage of the Hox B8 mRNA (Yekta et al
2004, Science 304-594). Otherwise, such interactions are known only in
plants (Bartel & Bartel 2003, Plant Physiol 132-709).

[0110] A number of studies have looked at the base-pairing requirement
between miRNA and its mRNA target for achieving efficient inhibition of
translation (reviewed by Bartel 2004, Cell 116-281). In mammalian cells,
the first 8 nucleotides of the miRNA may be important (Doench & Sharp
2004 GenesDev 2004-504). However, other parts of the microRNA may also
participate in mRNA binding. Moreover, sufficient base pairing at the 3'
can compensate for insufficient pairing at the 5' (Brennecke et al, 2005
PLoS 3-e85). Computation studies, analyzing miRNA binding on whole
genomes have suggested a specific role for bases 2-7 at the 5' of the
miRNA in target binding but the role of the first nucleotide, found
usually to be "A" was also recognized (Lewis et al 2005 Cell 120-15).
Similarly, nucleotides 1-7 or 2-8 were used to identify and validate
targets by Krek et al (2005, Nat Genet 37-495).

[0111] The target sites in the mRNA may be in the 5' UTR, the 3' UTR or in
the coding region. Interestingly, multiple miRNAs may regulate the same
mRNA target by recognizing the same or multiple sites. The presence of
multiple miRNA binding sites in most genetically identified targets may
indicate that the cooperative action of multiple RISCs provides the most
efficient translational inhibition.

[0112] MiRNAs may direct the RISC to downregulate gene expression by
either of two mechanisms: mRNA cleavage or translational repression. The
miRNA may specify cleavage of the mRNA if the mRNA has a certain degree
of complementarity to the miRNA. When a miRNA guides cleavage, the cut
may be between the nucleotides pairing to residues 10 and 11 of the
miRNA. Alternatively, the miRNA may repress translation if the miRNA does
not have the requisite degree of complementarity to the miRNA.
Translational repression may be more prevalent in animals since animals
may have a lower degree of complementarity between the miRNA and binding
site.

[0113] It should be noted that there may be variability in the 5' and 3'
ends of any pair of miRNA and miRNA*. This variability may be due to
variability in the enzymatic processing of Drosha and Dicer with respect
to the site of cleavage. Variability at the 5' and 3' ends of miRNA and
miRNA* may also be due to mismatches in the stem structures of the
pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a
population of different hairpin structures. Variability in the stem
structures may also lead to variability in the products of cleavage by
Drosha and Dicer.

Nucleic Acid

[0114] Nucleic acids are provided herein. The nucleic acid may comprise
the sequence of SEQ ID NOS: 1-110 or variants thereof. The variant may be
a complement of the referenced nucleotide sequence. The variant may also
be a nucleotide sequence that is substantially identical to the
referenced nucleotide sequence or the complement thereof. The variant may
also be a nucleotide sequence which hybridizes under stringent conditions
to the referenced nucleotide sequence, complements thereof, or nucleotide
sequences substantially identical thereto.

[0115] The nucleic acid may have a length of from 10 to 250 nucleotides.
The nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,
50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides. The
nucleic acid may be synthesized or expressed in a cell (in vitro or in
vivo) using a synthetic gene described herein. The nucleic acid may be
synthesized as a single strand molecule and hybridized to a substantially
complementary nucleic acid to form a duplex. The nucleic acid may be
introduced to a cell, tissue or organ in a single- or double-stranded
form or capable of being expressed by a synthetic gene using methods well
known to those skilled in the art, including as described in U.S. Pat.
No. 6,506,559 which is incorporated by reference.

[0116] Nucleic Acid Complexes

[0117] The nucleic acid may further comprise one or more of the following:
a peptide, a protein, a RNA-DNA hybrid, an antibody, an antibody
fragment, a Fab fragment, and an aptamer.

[0118] Pri-miRNA

[0119] The nucleic acid may comprise a sequence of a pri-miRNA or a
variant thereof. The pri-miRNA sequence may comprise from 45-30,000,
50-25,000, 100-20,000, 1,000-1,500 or 80-100 nucleotides. The sequence of
the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth
herein, and variants thereof. The sequence of the pri-miRNA may comprise
the sequence of SEQ ID NOS: 1-110 or variants thereof.

[0120] The pri-miRNA may form a hairpin structure. The hairpin may
comprise a first and second nucleic acid sequence that are substantially
complimentary. The first and second nucleic acid sequence may be from
37-50 nucleotides. The first and second nucleic acid sequence may be
separated by a third sequence of from 8-12 nucleotides. The hairpin
structure may have a free energy less than -25 Kcal/mole as calculated by
the Vienna algorithm with default parameters, as described in Hofacker et
al., Monatshefte f. Chemie 125: 167-188 (1994), the contents of which are
incorporated herein. The hairpin may comprise a terminal loop of 4-20,
8-12 or 10 nucleotides. The pri-miRNA may comprise at least 19% adenosine
nucleotides, at least 16% cytosine nucleotides, at least 23% thymine
nucleotides and at least 19% guanine nucleotides.

[0121] Pre-miRNA

[0122] The nucleic acid may also comprise a sequence of a pre-miRNA or a
variant thereof. The pre-miRNA sequence may comprise from 45-90, 60-80 or
60-70 nucleotides. The sequence of the pre-miRNA may comprise a miRNA and
a miRNA* as set forth herein. The sequence of the pre-miRNA may also be
that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3'
ends of the pri-miRNA. The sequence of the pre-miRNA may comprise the
sequence of SEQ ID NOS: 1-110 or variants thereof.

[0123] MiRNA

[0124] The nucleic acid may also comprise a sequence of a miRNA (including
miRNA*) or a variant thereof. The miRNA sequence may comprise from 13-33,
18-24 or 21-23 nucleotides. The miRNA may also comprise a total of at
least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40
nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides
of the pre-miRNA. The sequence of the miRNA may also be the last 13-33
nucleotides of the pre-miRNA. The sequence of the miRNA may comprise the
sequence of SEQ ID NOS: 1-25, 51-110 or variants thereof.

[0125] Anti-miRNA

[0126] The nucleic acid may also comprise a sequence of an anti-miRNA that
is capable of blocking the activity of a miRNA or miRNA*, such as by
binding to the pri-miRNA, pre-miRNA, miRNA or miRNA* (e.g. antisense or
RNA silencing), or by binding to the target binding site. The anti-miRNA
may comprise a total of 5-100 or 10-60 nucleotides. The anti-miRNA may
also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the anti-miRNA
may comprise (a) at least 5 nucleotides that are substantially identical
or complimentary to the 5' of a miRNA and at least 5-12 nucleotides that
are substantially complimentary to the flanking regions of the target
site from the 5' end of the miRNA, or (b) at least 5-12 nucleotides that
are substantially identical or complimentary to the 3' of a miRNA and at
least 5 nucleotide that are substantially complimentary to the flanking
region of the target site from the 3' end of the miRNA. The sequence of
the anti-miRNA may comprise the compliment of SEQ ID NOS: 1-110 or
variants thereof.

[0129] A synthetic gene is also provided comprising a nucleic acid
described herein operably linked to a transcriptional and/or
translational regulatory sequence. The synthetic gene may be capable of
modifying the expression of a target gene with a binding site for a
nucleic acid described herein. Expression of the target gene may be
modified in a cell, tissue or organ. The synthetic gene may be
synthesized or derived from naturally-occurring genes by standard
recombinant techniques. The synthetic gene may also comprise terminators
at the 3'-end of the transcriptional unit of the synthetic gene sequence.
The synthetic gene may also comprise a selectable marker.

Vector

[0130] A vector is also provided comprising a synthetic gene described
herein. The vector may be an expression vector. An expression vector may
comprise additional elements. For example, the expression vector may have
two replication systems allowing it to be maintained in two organisms,
e.g., in one host cell for expression and in a second host cell (e.g.,
bacteria) for cloning and amplification. For integrating expression
vectors, the expression vector may contain at least one sequence
homologous to the host cell genome, and preferably two homologous
sequences which flank the expression construct. The integrating vector
may be directed to a specific locus in the host cell by selecting the
appropriate homologous sequence for inclusion in the vector. The vector
may also comprise a selectable marker gene to allow the selection of
transformed host cells.

A probe may be capable of binding to a target nucleic acid of
complementary sequence through one or more types of chemical bonds,
usually through complementary base pairing, usually through hydrogen bond
formation. Probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the stringency of
the hybridization conditions. A probe may be single stranded or partially
single and partially double stranded. The strandedness of the probe is
dictated by the structure, composition, and properties of the target
sequence. Probes may be directly labeled or indirectly labeled.

Test Probe

[0133] The probe may be a test probe. The test probe may comprise a
nucleic acid sequence that is complementary to a miRNA, a miRNA*, a
pre-miRNA, or a pri-miRNA. The sequence of the test probe may be selected
from SEQ ID NOS: 1-110 or fragment thereof.

Linker Sequences

[0134] The probe may further comprise a linker. The linker may be 10-60
nucleotides in length. The linker may be 20-27 nucleotides in length. The
linker may be of sufficient length to allow the probe to be a total
length of 45-60 nucleotides. The linker may not be capable of forming a
stable secondary structure, may not be capable of folding on itself, or
may not be capable of folding on a non-linker portion of a nucleic acid
contained in the probe. The sequence of the linker may not appear in the
genome of the animal from which the probe non-linker nucleic acid is
derived.

Reverse Transcription

[0135] Target sequences of a cDNA may be generated by reverse
transcription of the target RNA. Methods for generating cDNA may be
reverse transcribing polyadenylated RNA or alternatively, RNA with a
ligated adaptor sequence.

Reverse Transcription Using Adaptor Sequence Ligated to RNA

[0136] The RNA may be ligated to an adapter sequence prior to reverse
transcription. A ligation reaction may be performed by T4 RNA ligase to
ligate an adaptor sequence at the 3' end of the RNA. Reverse
transcription (RT) reaction may then be performed using a primer
comprising a sequence that is complementary to the 3' end of the adaptor
sequence.

Reverse Transcription Using Polyadenylated Sequence Ligated to RNA

[0137] Polyadenylated RNA may be used in a reverse transcription (RT)
reaction using a poly(T) primer comprising a 5' adaptor sequence.

RT-PCR of RNA

[0138] The reverse transcript of the RNA may be amplified by real time
PCR, using a specific forward primer comprising at least 15 nucleic acids
complementary to the target nucleic acid and a 5' tail sequence; a
reverse primer that is complementary to the 3' end of the adaptor
sequence; and a probe comprising at least 8 nucleic acids complementary
to the target nucleic acid. The probe may be partially complementary to
the 5' end of the adaptor sequence.

PCR of Target Nucleic Acids

[0139] Methods of amplifying target nucleic acids are described herein.
The amplification may be by a method comprising PCR. The first cycles of
the PCR reaction may have an annealing temp of 56° C., 57°
C., 58° C., 59° C., or 60° C. The first cycles may
comprise 1-10 cycles. The remaining cycles of the PCR reaction may be
60° C. The remaining cycles may comprise 2-40 cycles. The
annealing temperature may cause the PCR to be more sensitive. The PCR may
generate longer products that can serve as higher stringency PCR
templates.

Forward Primer

[0140] The PCR reaction may comprise a forward primer. The forward primer
may comprise 15, 16, 17, 18, 19, 20, or 21 nucleotides identical to the
target nucleic acid. The 3' end of the forward primer may be sensitive to
differences in sequence between a target nucleic acid and a sibling
nucleic acid.

[0141] The forward primer may also comprise a 5' overhanging tail. The 5'
tail may increase the melting temperature of the forward primer. The
sequence of the 5' tail may comprise a sequence that is non-identical to
the genome of the animal from which the target nucleic acid is isolated.
The sequence of the 5' tail may also be synthetic. The 5' tail may
comprise 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides.

Reverse Primer

[0142] The PCR reaction may comprise a reverse primer. The reverse primer
may be complementary to a target nucleic acid. The reverse primer may
also comprise a sequence complementary to an adaptor sequence. The
sequence complementary to an adaptor sequence may comprise 12-24
nucleotides.

Biochip

[0143] A biochip is also provided. The biochip may comprise a solid
substrate comprising an attached probe or plurality of probes described
herein. The probes may be capable of hybridizing to a target sequence
under stringent hybridization conditions. The probes may be attached at
spatially defined addresses on the substrate. More than one probe per
target sequence may be used, with either overlapping probes or probes to
different sections of a particular target sequence. The probes may be
capable of hybridizing to target sequences associated with a single
disorder appreciated by those in the art. The probes may either be
synthesized first, with subsequent attachment to the biochip, or may be
directly synthesized on the biochip.

[0144] The solid substrate may be a material that may be modified to
contain discrete individual sites appropriate for the attachment or
association of the probes and is amenable to at least one detection
method. Representative examples of substrates include glass and modified
or functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene, polyethylene,
polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
plastics. The substrates may allow optical detection without appreciably
fluorescing.

[0145] The substrate may be planar, although other configurations of
substrates may be used as well. For example, probes may be placed on the
inside surface of a tube, for flow-through sample analysis to minimize
sample volume. Similarly, the substrate may be flexible, such as a
flexible foam, including closed cell foams made of particular plastics.

[0146] The biochip and the probe may be derivatized with chemical
functional groups for subsequent attachment of the two. For example, the
biochip may be derivatized with a chemical functional group including,
but not limited to, amino groups, carboxyl groups, oxo groups or thiol
groups. Using these functional groups, the probes may be attached using
functional groups on the probes either directly or indirectly using a
linker. The probes may be attached to the solid support by either the 5'
terminus, 3' terminus, or via an internal nucleotide.

[0147] The probe may also be attached to the solid support non-covalently.
For example, biotinylated oligonucleotides can be made, which may bind to
surfaces covalently coated with streptavidin, resulting in attachment.
Alternatively, probes may be synthesized on the surface using techniques
such as photopolymerization and photolithography.

Diagnostic

[0148] A method of diagnosis is also provided. The method comprises
detecting a differential expression level of preeclampsia or preterm
labor associated nucleic acid in a biological sample. The sample may be
derived from a female patient. Diagnosis of preeclampsia or preterm labor
in a female patient may allow for prognosis and selection of therapeutic
strategy. The skilled artisan can make a diagnosis, a prognosis, or a
prediction based on the findings.

Kits

[0149] A kit is also provided and may comprise a nucleic acid described
herein together with any or all of the following: assay reagents,
buffers, probes and/or primers, and sterile saline or another
pharmaceutically acceptable emulsion and suspension base. In addition,
the kits may include instructional materials containing directions (e.g.,
protocols) for the practice of the methods described herein.

[0150] For example, the kit may be a kit for the amplification, detection,
identification or quantification of a target nucleic acid sequence. The
kit may comprise a poly (T) primer, a forward primer, a reverse primer,
and a probe.

[0151] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no way be
construed, however, as limiting the broad scope of the invention.

EXAMPLES

Example 1

Experimental Procedures

[0152] 1. Study Population

[0153] The first study group included 20 pregnant women: 10 in the first
trimester (6-12 weeks of gestational age) and 10 in the third trimester
(34-41 weeks of gestational age) and from 10 control, age-matched
non-pregnant women. Eligibility for the study was limited to normal
uncomplicated singleton pregnancy with no known fetal malformation. All
women provided written informed consent and the local institutional
review board approved the study.

[0154] The second study group included women with preeclampsia that were
delivered by cesarean section prior to onset of spontaneous delivery. The
control group included women delivering by cesarean section prior to
onset of spontaneous delivery with no clinical or laboratory evidence of
hypertensive disorder of pregnancy. Spontaneous onset of labor was
defined by regular painful contractions or cervical dilatation>=4 cm.
Preeclampsia was defined based on elevated blood pressure
(systolic>=140 mmHg or diastolic>=90 mmHg) and proteinuria
(>=300 mg/24 h or >=1+dipstick) that appear after 20 weeks of
gestation. Severe preeclampsia was defined as preeclampsia with one of
the following: systolic blood pressure>=160 mmHg, diastolic blood
pressure>=110 mmHg, proteinuria>=5 grams/24 h, the presence of
headache, visual disturbance or persistent epigastric pain, seizures,
oliguria, elevated creatinine levels, platelets count<100,000/uL,
elevated liver enzymes, evidence of hemolysis, or fetal growth
restriction according to local birthweight curves. Exclusion criteria
included multiple gestations, women with chronic hypertension,
gestational age<24 weeks, uncertain gestational age, or clinical or
histological evidence of chorioamnionitis. The third study group included
women with spontaneous preterm onset of delivery (<34 weeks of
gestation), during active phase of delivery (>4 cm cervical dilatation
and regular contractions) and control group of women delivering at term
(women with suspected chorioamniotis were excluded). All women in the
current study were delivered by cesarean section. All participants
provided written informed consent and the local IRB committee approved
the study.

[0155] 2. Sampling of Body Fluids, Placenta and Myometrium

[0156] Prior to the onset of cesarean section, a blood sample (5 cc, in a
sodium citrate containing test-tube), was taken. During cesarean section,
after delivery of the infant, the placenta was manually removed. Full
thickness samples (about 1 g each) were taken from the placenta at areas
that macroscopically had no evidence of abruption or infarction. After
delivery of the infant and removal of the placenta, a full thickness
myometrial sample about 5×5 mm was taken from the superior edge of
the transverse uterine incision with a curved Mayo-scissors. Both
placental and myometrium samples were immediately frozen in liquid
nitrogen and transferred for storage in a -70° C. refrigerator.
Because previous studies have shown that there is a dramatic change in
gene expression profile in the placenta during labor, all samples were
taken from placentas of women undergoing cesarean section prior to the
onset of labor.

Serum Samples

[0157] 8 ml of blood was collected from each woman directly into serum
collection tubes (Greiner Bio-one, VACUETTE® Serum Tubes 455071). The
whole blood was allowed to stand for about 1 h at RT before being
centrifuged at 1800 g for 10 minutes at RT. The resultant serum was
aliquoted into eppendorf tubes and stored at -80° C.

Urine Samples

[0158] About 4 ml of urine was collected from each individual in a urine
container. The urine was then aliquoted into eppendorf tubes and kept
frozen at -80° C. until it was used for RNA extraction.

[0159] 3. miR Microarray Platform

[0160] Custom microarrays were produced by printing DNA oligonucleotide
probes representing 688 miRNAs [Sanger database, version 9.2 (miRBase:
microRNA sequences, targets and gene nomenclature. Griffiths-Jones S,
Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,
Database Issue, D140-D144) and additional Rosetta genomics validated and
predicted miRs]. Each probe carries up to 22-nt linker at the 3' end of
the miRNA's complement sequence in addition to an amine group used to
couple the probes to coated glass slides. 20 μM of each probe were
dissolved in 2×SSC+0.0035% SDS and spotted in triplicate on Schott
Nexterion® Slide E coated microarray slides using a Genomic
Solutions® BioRobotics MicroGrid II according the MicroGrid
manufacturer's directions. 64 negative control probes were designed using
the sense sequences of different miRNAs. Two groups of positive control
probes were designed to hybridize to miR microarray. Synthetic spikes
small RNA were added to the RNA before labeling to verify the labeling
efficiency and (2) probes for abundant small RNA (e.g. small nuclear RNAs
(U43, U49, U24, Z30, U6, U48, U44), 5.8s and 5s ribosomal RNA) were
spotted on the array to verify RNA quality. The slides were blocked in a
solution containing 50 mM ethanolamine, 1M Tris (pH 9.0) and 0.1% SDS for
20 min at 50° C., then thoroughly rinsed with water and spun dry.

[0161] 4. Cy-Dye Labeling of microRNA for miR Microarray

[0162] 1.5-3.5 μg of total RNA was labeled by ligation of a RNA-linker
p-rCrU-Cy- dye (Thomson et al., 2004, Nat Methods 1, 47-53) (Eurogentec)
to the 3'-end with Cy3 or Cy5. The labeling reaction contained total RNA,
spikes (20-0.1 fmoles), 500 ng RNA-linker-dye, 15% DMSO, 1× ligase
buffer and 20 units of T4 RNA ligase (NEB) and proceeded at 4° C.
for 1 hr followed by 1 hr at 37° C. The labeled RNA was mixed with
3× hybridization buffer (Ambion), heated to 95° C. for 3 min
and than added on top of the miR microarray. Slides were hybridize 12-16
hr, followed by two washes with 1×SSC and 0.2% SDS and a final wash
with 0.1×SSC.

[0163] The array was scanned using an Agilent Microarray Scanner Bundle
G2565BA (resolution of 10 μm at 100% power). The data was analyzed
using SpotReader software.

[0164] 5. RNA Extraction

[0165] RNA was extracted from frozen samples originated from placental
tissue and myometrium. Total RNA from frozen tissues was extracted with
the miRvana miRNA isolation kit (Ambion) according to the manufacturer's
instructions.

[0166] 100 ul serum or urine was incubated at 56° C. for 1 h with
0.65 mg/ml Proteinase K, (Sigma P2308). Two synthetic RNAs were spiked-in
as controls before acid phenol:chloroform extraction and then RNA was
ETOH precipitated ON at -20° C. Next, DNase treatment was
performed to eliminate residual DNA fragments. Finally, after a second
acid phenol:chloroform extraction, the pellet was re-suspended in DDW and
two additional synthetic RNAs are spiked-in as controls.

[0167] 6. miR qRT-PCR Platform

[0168] RNA was subjected to polyadenylation reaction as described
previously (Rui Shi and Vincent L. Chiang. Facile means for quantifying
microRNA expression by real-time PCR. BioTechniques (2005) 39:519-525).
Briefly, RNA was incubated in the presence of poly (A) polymerase (PAP)
(Takara-2180A), PNK buffer (NEB) MnCl2, and ATP for 1 h at
37° C. Then, using an oligodT primer harboring a consensus
sequence (complementary to the reverse primer) reverse transcription was
performed on total RNA, using SuperScript II RT (Invitrogen).

[0169] Next, the cDNA was amplified by real time PCR; this reaction
contained a microRNA-specific forward primer, a TaqMan probe
complementary to 8 nts of the 3' end of the specific microRNA sequence as
well as to 12 nts of the polyA adaptor and to few bases (2-4) on the 5'
of the oligodT tail; and universal reverse primer complementary to the 3'
sequence of the oligo dT tail.

Example 2

microRNAs in Body Fluids Represent Novel Clinical Biomarkers

[0170] We have developed a protocol for extracting cell-free microRNAs
from body fluids (see Example 1). Assessment of extracted microRNA levels
was achieved using a proprietary qRT-PCR technique, which is highly
sensitive. The qRT-PCR method detects specifically mature microRNA
molecules, and allows discrimination between homologous microRNA family
members that differ by a single nucleotide. The sensitivity and
specificity of this qRT-PCR method is demonstrated by our ability to
detect a few molecules of microRNA present in a non-relevant RNA
background. Such high sensitivity makes it possible to use qRT-PCR to
monitor the minute amount of microRNA present in cell-free body fluids.

[0171] In order for microRNAs in serum to be useful biomarkers they must
be stable for reasonable periods of time to allow for routine processing
of clinical samples. We found that the expression levels of different
microRNAs in unfrozen serum do not change substantially over a 4 hour
period at room temperature, and also are not affected by twice freezing
and re-thawing of samples (data not shown). Thus, microRNAs in serum are
sufficiently robust to serve as potential clinical biomarkers.
Additionally, we observed that these microRNAs are expressed similarly in
serum samples taken from different healthy individuals. Therefore, we
anticipate that differences in expression between individuals of only
particular microRNAs could be used to indicate clinical conditions.
Notably, using our extraction and qRT-PCR methods we established that
microRNAs are also present in other body fluids, such as urine, saliva,
amniotic fluid and pleural fluid.

[0172] Finally, as a proof of concept, we investigated whether circulating
microRNAs can be used to identify clinical conditions. It has been
established that circulating maternal RNA contains placental embryonic
RNA. We measured the levels of 28 microRNAs, including placenta-specific
microRNAs, as well as broadly expressed microRNAs.

[0173] The median fold changes in microRNA levels comparing third
trimester pregnant women to non pregnant women are detailed in Table 1.
Box plots show relative microRNA expression levels in the sera of 10 non
pregnant women, 10 women in the first trimester and 10 women in the third
trimester (FIG. 1A). Hsa-miR-526a (SEQ ID NO: 75) and hsa-miR-527 (SEQ ID
NO: 53) are upregulated dramatically in the serum of third trimester
pregnant women (more than 600 fold), and the expression levels of several
other microRNAs are also significantly increased during pregnancy (Table
1 and FIG. 1A). The expression levels of the placental microRNAs rise
with gestational age (FIG. 1A). Indeed, we found that the expression
levels of three placental microRNAs (hsa-miR-526a, hsa-miR-527 and
hsa-miR-520d-5p) could be used to accurately distinguish pregnant from
non pregnant women (FIG. 1C) and even to identify different stages of
pregnancy.

[0174] We have developed highly sensitive methods that enable the
extraction and measurement of cell-free microRNAs in body fluids. Here,
we establish that microRNAs are indeed present in serum and in other body
fluids. We show that microRNA levels in serum are consistent across
individuals and stable during routine processing of clinical samples.
Importantly, we demonstrate that certain microRNAs in serum are expressed
differentially under dissimilar physiological conditions, namely during
pregnancy. Thus, circulating microRNAs represent promising candidates for
robust, sensitive and easily accessible biomarkers.

[0175] For each microRNA, "delta CT" indicates the difference in
median CT between the serum of pregnant women in the third trimester
(n=10) and non-pregnant women (n=10). For each sample, the relative
amount of the microRNAs was normalized by subtracting the average CT
of the non-placenta-specific microRNAs. The fold change is the ratio of
the median abundance in linear space, equal to the exponent (base 2) of
the delta CT. P-values are calculated by a two-sided unpaired
t-test.

Example 3

Differential Expression of microRNAs in Placenta and Serum Samples
Obtained from Women with Severe Preeclampsia (PET) and Healthy Women

[0176] Overall 33 female patients were evaluated, among them 15 with
severe PET and 18 served as control. Significant difference in microRNAs
expression profile was found in placentas derived from patients with PET
in comparison to the control group.

[0179] These findings suggest that specific microRNAs may play an
essential role in the pathogenesis and diagnosis of PET.

Example 4

Differential Expression of microRNAs in Specimen of Uterine Myometrium and
Placenta Obtained from Women with Spontaneous Preterm Onset of Delivery
(sPTL) and from Control Group

[0180] Overall 10 female patients were evaluated, among them 5 with
spontaneous preterm onset of delivery and 5 women delivering at term
served as control. Significant difference in microRNAs expression
profiles was found in placentas and uterine myometrium derived from
patients with sPTL in comparison to the control group.

[0181] As shown in FIG. 10, hsa-miR 210 (SEQ ID NO: 18) and hsa-miR-223
(SEQ ID NO: 19) are differentially expressed in specimen of uterine
myometrium obtained from women with sPTL in comparison to control group.